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Flavour encapsulation in milk proteins – CMC coacervate-type complexes
Food Hydrocolloids 37 (2014) 134e142
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Flavour encapsulation in milk proteins e CMC coacervate-type complexes T. Koupantsis a, E. Pavlidou b, A. Paraskevopoulou a, * a b
Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece Solid State Physics Section, Physics Department, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2013 Accepted 31 October 2013
Beta-pinene containing microcapsules were prepared by complex coacervation of milk proteins, i.e sodium caseinate (CN) and whey protein isolate (WPI), with carboxymethylcellulose (CMC). Milk proteins e CMC interactions were followed by z-potential measurements, while the initial emulsions were characterised for droplet size and biopolymer amount present at the oil/water interface. Response surface methodology was applied to investigate the effects of encapsulation processing variables, including protein/polysaccharide (pr/pl) ratio and volatile compound’s mass, on encapsulation yield, loading and efficiency as well as the morphological characteristics of the produced microcapsules. The obtained results revealed that it was possible to encapsulate b-pinene with milk proteins and CMC by complex coacervation, while most of the characteristics evaluated were affected by the process variables. Coacervates prepared at the highest pr/pl ratio of 6.99 and b-pinene mass (6.99 g) were the most effective in encapsulating the flavour compound, something that was more evident in the case of WPIeCMC mixture. Additionally, microcapsule structure, evaluated by Scanning Electron Microscopy analysis, was noticeably affected by the protein/polysaccharide ratio being compact when pr/pl was low and “spongy”like when it was high. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Flavour encapsulation Complex coacervation Sodium caseinate Whey protein Carboxymethylcellulose Response surface methodology
1. Introduction Proteinepolysaccharide interactions have found diverse applications in food sector, such as in food colloidal systems formation, fat substitution, protein recovery from fluid by-products and encapsulation (Damianou & Kiosseoglou, 2006; Dickinson, 2003; Kika, Korlos, & Kiosseoglou, 2007; Paraskevopoulou & Kiosseoglou, 2013). Among them, biopolymer-based encapsulation of various ingredients (flavours, vitamins, antioxidants, etc.) offers many advantages in view of protection from losses and environmental factors, unpleasant tastes’ masking, liquid conversion to more convenient solid materials and controlled release (Nori et al., 2011; Qv, Zeng, & Jiang, 2011; Zhang, Pan, & Chung, 2011). In the case of flavour compounds, encapsulation provides an effective way for their stability reinforcement through elimination of problems associated with their exposure to air, light, heat and moisture. It is based on the formation of a coating layer (wall material) to encapsulate flavour droplets or particles (core material). Proteinepolysaccharide complex coacervation is the process
* Corresponding author. Tel.: þ30 2 31 0 997832; fax: þ30 231 0 997847, 997779. E-mail address: [email protected] (A. Paraskevopoulou). 0268-005X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2013.10.031
most commonly used for this purpose. It is, almost entirely, accomplished when two oppositely charged biopolymer molecules are mixed at a pH below the protein isoelectric point (pI) leading to the formation of a complex, i.e. coacervate, which precipitates retaining at the same time the aroma material at the coacervate phase. The success of the process is mainly affected by pH manipulation. More specifically, a soluble proteinepolysaccharide complex is firstly formed followed by complex coacervation and/or precipitation upon further lowering of pH, attributed to the modulation of initial repulsive proteinepolysaccharide interactions into net attractive ones (Paraskevopoulou & Kiosseoglou, 2013). Flavour encapsulation by complex coacervation of proteins with a number of charge-carrying polysaccharides has been the subject of some recently published research papers (Jun-xia, Hai-yan, & Jian, 2011; Leclercq, Harlander, & Reineccius, 2009; Prata, Zanin, Ré, & Grosso, 2008; Yeo, Bellas, Firestone, Langer, & Kohane, 2005). In these studies, various combinations including gelatine or soybean protein isolate with gum arabic, xanthan or pectin have been used for the encapsulation of essential oils or individual flavour compounds. In the present study milk proteins, i.e. sodium caseinate and whey protein isolate, were employed in admixture with carboxymethylcellulose. Milk proteins are commonly used in the food industry for their excellent functional (emulsion preparation and
T. Koupantsis et al. / Food Hydrocolloids 37 (2014) 134e142
stabilisation, water and fat binding, thickening, gelation) and nutritional properties. CN is composed of a mixture of four phosphoproteins, namely as1-, as2-, b- and k-casein. Their highly disordered and hydrophobic character is considered to be responsible for sodium caseinate’s rapid absorption to the oilewater interface during emulsification as well as stabilisation of oil droplets against flocculation and coalescence (Dickinson, 1997). The major component of WPI is b-lactoglobulin, a globular protein known to interact with many aroma compounds due to the existence on the molecule of two independent binding sites (Narayan & Berliner, 1997). Carboxymethylcellulose was selected because it is an anionic polysaccharide that is already widely used as a functional ingredient by the food industry. Its manufacture involves treating cellulose with aqueous sodium hydroxide followed by reaction with monochloroacetic acid. It is water soluble and its solutions exhibit non-Newtonian, pseudoplastic solutions (Zecher & Gerrish, 1999). Milk proteins have been successfully used in combination with polysaccharides such as gum arabic, xanthan, mesquite gum as well as CMC in the formation and stabilisation of food emulsion systems, while, depending on pH, both of them interact with polysaccharides to form soluble or insoluble complex coacervates, nanoparticles or precipitates (Bedie, Turgeon, & Makhlouf, 2008; Benichou, Aserin, Lutz, & Garti, 2007; Ercelebi & Ibanoglu, 2007; Koupantsis & Kiosseoglou, 2009; Liu et al., 2012; Weinbreck, Minor, & de Kruif, 2004; Ye, 2008). More specifically, it was observed that when the pH of an aqueous WPIeCMC solution was steadily reduced to values between 3.5 and 4, a gel-like system was generated upon ageing (Koupantsis & Kiosseoglou, 2009; Paraskevopoulou, Tsioga, Biliaderis, & Kiosseoglou, 2013). The development of the gel structure was primarily attributed to electrostatic interactions between the two biopolymers, while hydrophobic interactions between protein molecules appeared to play a minor role. Additionally, the gel structure formation at low pH enhanced the retention of orange oil aroma compounds when compared with single biopolymers or their mixtures at neutral pH (Paraskevopoulou et al., 2013). In view of the above, this work was conducted to investigate whether complex coacervation of milk proteins with CMC can be used for the encapsulation of flavour compounds, i.e. b-pinene. The initial emulsions were characterised for droplet size and biopolymer adsorption to the oil/water interface, while the microcapsules were characterised for encapsulation efficiency, loading, yield and morphology. Optimisation of the encapsulation process conditions was attempted using response surface methodology where the simultaneous effect of b-pinene amount and protein/polysaccharide mass ratio was investigated. 2. Materials and methods 2.1. Materials Casein sodium salt (from bovine milk) was purchased from Sigma Chemical Co. (Germany). Commercial whey protein isolate (natural, unflavoured, lecithin content <3%) from bovine milk was supplied by Nestlé (Germany). This product contained approximately 98% dry matter, of which 91% was protein. Carboxymethylcellulose sodium salt (with low viscosity of 90e200 mPa at 25 C) and b-pinene (>95%, CAS No.127-91-3) were purchased from Fluka (Switzerland). Analytical grade n-hexane was obtained from Merck (Germany). Sodium azide (Aldrich, Germany) was added to the protein and polysaccharide solutions to prevent microbial growth (final concentration of 0.01% w/v). Hydrochloric acid and sodium hydroxide solutions used for pH adjustment were purchased from Aldrich (Germany). Deionised water was used for the preparation of all solutions and emulsions.
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2.2. Microcapsule preparation 2.2.1. Preparation of biopolymer matrices Individual protein (3.5% w/v) and polysaccharide (0.5% w/v) matrices were initially prepared by dispersing sodium caseinate, whey protein isolate and carboxymethylcellulose in deionised water under mechanical stirring at room temperature for more than 5 h for a full dispersion of the macromolecules. The three solutions were then used to prepare the emulsions. Blends containing 0.25% (w/v) CMC and different amounts of CN or WPI were obtained by mechanical mixing at 300 rpm (IKA, Malaysia) for at least 30 min to assure the complete formation of biopolymer mixtures (Table 1). 2.2.2. Formation of o/w emulsions Oil-in-water emulsions were prepared at room temperature by mixing appropriate amount of b-pinene with 200 mL protein/polysaccharide solution with the aid of a mechanical stirrer at 600 rpm (IKA, Malaysia), followed by homogenisation at 30 106 Pa (4 passes) using an APV-2000 pressure homogenizer (APV Systems, Denmark). The produced emulsions showed a final composition, which is shown in Table 1. Sodium azide (0.01% w/v) was added to all emulsions in order to prevent microbial growth. The losses during emulsion preparation were found to be very limited (<2%) and were mainly due to emulsion quantity remaining in the glassware and apparatus used for its preparation as well as volatilisation in the air. 2.2.3. Preparation of microcapsules Four hundred millilitres of deionised water was slowly poured into a beaker containing the final emulsion (w200 mL) and the pH was adjusted to 2.8 with hydrochloric acid solution (1 mol/L) under continuous agitation with the aid of a magnetic stirrer (300 rpm). During the coacervation process the temperature was kept at around 23 C and the suspension was kept at w4 C overnight to allow the coacervation process to be completed (w20 h) (Lamprecht, Schafer, & Lehr, 2001; Leclercq et al., 2009; Zhang et al., 2011). The precipitates were first washed by decanting with deionised water and then isolated by centrifugation (4000 rpm, 10 min), frozen and lyophilised for 5e6 h by using a laboratoryscale freeze dryer (Alpha 1-2, Christ, Germany). The resulting microcapsules were stored at 4 C in a desiccator for further analyses. 2.3. Emulsion droplet size measurement Droplet size distributions and average droplet diameter D[3,2] of the freshly prepared emulsions (before pH adjustment) was determined by the laser diffraction technique and the aid of a Malvern Mastersizer 2000 (Malvern Instruments, Worcestershire, UK) (b-pinene and water refractive index: 1.4650 and 1.33, respectively; Absorption: 0.002) (Reich & Sanhueza, 1993). Each sample was analysed in triplicate and each replicate was measured twice to have the average particle size. 2.4. z-Potential measurements of protein and polysaccharide solutions The electrical charge of CN, WPI and CMC solutions and their mixtures was measured using a Zetasizer ZEN2600 (Malvern Instruments, Worcestershire, UK). For this reason, stock solutions of each biopolymer were first prepared (0.05% w/v). Mixed solutions containing CMC and CN or WPI at various ratios (Table 1) were obtained by thorough mixing appropriate amounts of the initial stock solutions. The individual as well as the mixed solutions were then diluted to a concentration of approximately 0.02% (w/v) with deionised water and, after adjustment to the suitable pH, were
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Table 1 Experimental design (conditions and responses) for encapsulation yield (EY%), encapsulation efficiency (EE%), encapsulation loading (EL%), adsorbed amount of biopolymers per surface unit (Gs) and emulsion droplet diameter (D[3,2]). xPr:yPla
Run
Factor A Pr/Pl ratio
Factor B
b-pinene (g)
CNeCMC EY (%)
WPIeCMC EE (%)
EL (%)
Gs (CN)
Gs (CMC)
EY (%)
(mg/m2)
D[3,2] (mm)
10.05 38.50 39.73 61.10 10.23 10.54 7.55 9.87 10.85 10.97 7.35 11.23 3.70
5.28 21.95 21.67 33.59 5.41 5.13 3.87 4.69 5.38 4.70 4.68 7.05 3.62
0.256 0.332 0.213 0.221 0.245 0.273 0.341 0.265 0.278 0.247 0.362 0.408 0.330
20.80 29.17 18.41 42.06 22.29 23.56 6.34 21.90 20.90 1.02 4.88 19.06 12.21
(mg/m2) 1 2 3 4 5 6 7 8 9 10 11 12 13 a
52:100 17:100 86:100 52:100 52:100 52:100 86:100 52:100 52:100 100:100 3:100 52:100 17:100
3.60 1.20 6.00 3.60 3.60 3.60 6.00 3.60 3.60 6.99 0.21 3.60 1.20
3.60 1.20 1.20 0.21 3.60 3.60 6.00 3.60 3.60 3.60 3.60 6.99 6.00
25.20 34.96 39.32 47.81 26.20 25.44 20.37 25.54 28.25 27.33 14.50 23.57 19.42
17.80 26.80 17.83 26.19 19.01 21.31 28.17 21.55 20.59 19.03 29.02 27.28 28.25
10.86 11.09 4.55 2.15 8.88 12.56 17.79 11.60 13.00 10.05 25.09 20.53 23.87
EE (%)
EL (%)
Gs (WPI)
Gs (CMC)
(mg/m2)
D[3,2] (mm)
10.63 47.15 36.05 69.80 9.31 11.83 5.37 8.79 11.27 5.57 7.53 8.92 2.75
5.40 43.56 24.65 50.61 5.69 5.54 2.60 5.15 5.05 3.23 13.50 2.77 0.27
0.260 0.560 0.165 0.310 0.288 0.300 0.291 0.307 0.281 0.240 0.490 0.299 0.357
(mg/m2) 24.95 22.00 22.07 13.05 26.11 23.57 36.90 28.67 28.58 28.33 35.54 30.44 26.39
13.39 13.67 5.64 1.07 15.93 14.38 23.31 17.49 17.44 11.12 30.10 22.91 22.30
“x” and “y”: volumes of initial protein (3.5% w/v) and polysaccharide (0.5% w/v) solutions, respectively.
placed into the measurement cell of the instrument. The measurements were converted to z-potential values by applying the Smoluchowski mathematical model and are reported as average of three replicates, each of which was measured three times. The temperature was kept constant at 22 C.
after it was taken out of the freeze, it was centrifuged at 4000 rpm for 40 min, placed in a water bath of 50 C for 40 min and recentrifuged at 4000 rpm for 10 min until complete phase separation. The organic phase was collected and was subsequently stored in screw-capped glass vials at 18 C until further analysis. Each powdered sample was extracted in triplicate.
2.5. Protein and polysaccharide surface amount determination The amount of protein and polysaccharide adsorbed at the surface of the emulsion droplets was determined from the difference between the initial amount of biopolymers used and that remained in the serum after the coacervation was completed. Total protein and polysaccharide content was determined by applying the FolineCiocalteu (Lowry, Rosebrough, Farr, & Randall, 1951) and phenolesulphuric method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), respectively. More specifically, for the determination of the surface protein (or polysaccharide) concentration Gs (mg/m2), the following equations were used (Walstra, 1983):
SSA ¼
6 D½3;2
ST ¼ SSA$V
GS ¼
GT
2.7. GC-FID analyses The analyses were accomplished with an Agilent 6890A gas chromatograph equipped with a split-splitless injector and a flame ionization detector (FID). The samples were analysed on a HP-FFAP column (25 m 0.20 mm i.d., film thickness 0.30 mm; Agilent Technologies). The carrier gas was helium at a constant flow rate of 1 mL/min. Samples (2 mL) were injected manually into the GC in split mode with 1:100 ratio. Injector and detector were both kept at 230 C. The temperature program was 40 C for 2 min, raised to 100 C at 10 C/min, then raised to 230 C at 30 C/min and held for 1 min. The obtained peak areas were converted to concentrations using a calibration curve (y ¼ 0.3732x þ 44.656). For its construction, b-pinene solutions were prepared in hexane at ten different concentration levels (0.10e6.24 g/L) and analysed five times applying the same conditions as described previously for the samples. Linear correlation coefficient was found equal to 0.9985. All analyses were performed at least three times (CV < 3%).
ST
where, SSA is the specific surface area (m2/mL), D[3,2] is the volumeesurface mean droplet diameter (mm), ST is the total surface of oil droplets (m2), V is the volume of b-pinene (mL) and GT is the total biopolymer at the surface (mg) calculated by the difference between the biopolymer amount used initially and that found in the serum. 2.6. Extraction of encapsulated b-pinene Beta-pinene was extracted as described by other researchers with slight modifications (Soottitantawat et al., 2004, 2005; Zhang et al., 2011). Five mL of deionised water was added to 0.1 g of the freeze-dried powder in a screw-capped glass tube, followed by 2 mL hexane (pH was adjusted to 7.0 by using a 0.1 mol/L sodium hydroxide solution). The resulting solution was vortexed for 1 min at room temperature and stored at 18 C overnight. Immediately
2.8. Microcapsules characterisation The b-pinene containing particles were characterised with respect to yield, encapsulation efficiency, encapsulation loading and morphology. The three encapsulation monitoring parameters were calculated as follows:
Encapsulation Yield ð%Þ ¼
Mmicrocapsules $100% Mtotal
Encapsulation Loading ð%Þ ¼
Encapsulation Efficiency ð%Þ ¼
Mbpinene encapsulated $100% Mmicrocapsules Mbpinene Mbpinene to
encapsulated be encapsulated
$100%
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where Mmicrocapsules is the mass of the final product, Mtotal is the mass of initial material used (wall and core), Mb-pinene encapsulated is the actual amount of b-pinene encapsulated and Mb-pinene to be encapsulated the theoretical mass of b-pinene to be encapsulated. The morphology of the microcapsules was performed by Scanning Electron Microscopy (SEM), using a JEOL 840A SEM operated at 20 kV equipped with an INCA 300 EDS analyzer. The dried samples under investigation were placed on sample holders and coated by carbon, using a JEE-4X vacuum evaporator, in order to achieve electrical conductivity.
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A
2.9. Experimental design A rotatable central composite design was used to perform the tests for the microencapsulation of b-pinene, considering two factors (independent variables): mass of b-pinene (0.21e6.99 g) and the mass ratio of protein (CN or WPI) and polysaccharide (CMC) (0.21e6.99 g). The experimental plan consisted of 13 trials (5 for the central point replicated for the estimation of error) and the independent variables were studied at five different levels (Table 1). Central point conditions were 3.6 g b-pinene and 3.6 protein/ polysaccharide mass ratio. The following polynomial equation was fitted to data:
B
y ¼ b0 þ b1 A þ b2 B þ b11 A2 þ b22 B2 þ b12 AB where, bn are constant regression coefficients, y is the response variable (encapsulation efficiency, encapsulation loading or encapsulation yield) and A and B are the coded independent variables corresponding to protein/polysaccharide mass ratio and bpinene mass, respectively. The statistical analysis of the data was performed by using MINITABÔ Statistical Software and the level of significance was 95%. The quality of fit of the model was evaluated on the basis of coefficients of determination (R2), the significance of each parameter (through calculated p values) and the “lack of fit”.
C
3. Results and discussion 3.1. pH selection In this study, because of the use of the anionic polysaccharide carboxymethylcellulose along with sodium caseinate or whey protein isolate, complex coacervation was found to be favoured at a pH around 2.8, significantly below the protein isoelectric point (w4.6 for CN and 4.1e5.2 for WPI) (Koupantsis & Kiosseoglou, 2009; Liu et al., 2012). For the selection of this pH, z-potential values (Fig. 1a) as well the mass of the precipitated material and the turbidity of the supernatant (data not shown) at various pH levels (range 2.5e7.3) were taken into account (Nori et al., 2011). More specifically, as Fig. 1a reveals, at pH 2.8 there is enough charge in order interactions between protein and polysaccharide to take place. In addition, at this pH value the supernatant was almost a clear solution while the larger quantity of precipitate was observed. Generally, in the case of anionic polysaccharides the optimum pH for coacervation is below the protein isoelectric point (pI) which is explained by the development of intense attractive electrostatic forces between the biopolymers (Koupantsis & Kiosseoglou, 2009; Paraskevopoulou et al., 2013; Paraskevopoulou & Kiosseoglou, 2013). Generally, at a pH close to neutrality (w6.8) interactions between protein and polysaccharide molecules were not favoured as both of them carry a net negative charge (Fig. 1b). However, the existence of weak interactions has been reported by several researchers for various proteinepolysaccharide pairs, such as BSAeicarrageenan, WPIexanthan, WPC or WPIeCMC (Benichou et al.,
Fig. 1. z-potential of biopolymer solutions as affected by the pH (A) and the protein/ polysaccharide ratio at pH 6.8 (B) and at pH 2.8 (C). Keys: CN; WPI; CMC; CNe CMC mixture; WPIeCMC mixture.
2007; Koupantsis & Kiosseoglou, 2009; Paraskevopoulou et al., 2013). The emulsions prepared with the use of both studied combinations (CNeCMC and WPIeCMC) exhibited monomodal distribution and similar droplet mean diameter characteristics. As pH was decreased to a value of 2.8, the protein molecules became positively charged while CMC molecules less negatively charged (Fig. 1a). This suggests that, upon lowering of pH to a value below enough the isoelectric point of the proteins, electrostatic interactions between positively and negatively charges located on the protein and CMC chains, respectively, took place which led to the formation of insoluble complexes (Paraskevopoulou et al., 2013; Schmitt & Turgeon, 2011; Ye, 2008). As Fig. 1c shows, at this pH the biopolymers possessed sufficient electric charge that allowed the molecules to interact with each other. Moreover, the biopolymer mixing ratio is considered to be crucial in controlling system’s charge and thus the extent of interactions and aggregation
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phenomena between protein and polysaccharide (Ye, 2008). Actually, depending on the protein/CMC ratio, these dispersed proteine polysaccharide coacervates aggregated and formed clusters that grew in size and, in most of the cases, precipitated (visually observed). This is in agreement with the reports of many researchers for various proteinepolysaccharide systems, including soybean protein isolateepectin and gelatineegum arabic, who indicated that at a pH between the protein pI and polysaccharide pK, this interaction, which is mainly electrostatic in nature, usually results in biopolymer coacervation and, upon further lowering of pH, in insoluble complex formation and precipitation (Nori et al., 2011; Leclercq et al., 2009; Zhang et al., 2011). In addition to electrostatic interactions, physical interactions, such as hydrogen and/ or hydrophobic ones, may play a role in proteinepolysaccharide coacervation and complex formation (Tolstoguzov, 1986). 3.2. Response surface methodology The process of b-pinene-containing microcapsules production was standardised for the maximum encapsulation by using response surface methodology. As has already been mentioned, two different biopolymer combinations were tested, i.e. CMC was combined either with CN or WPI by using complex coacervation. Three parameters were applied for the characterisation of dried microcapsules, namely yield of the encapsulation procedure (EY), encapsulation loading of b-pinene (EL) in the final product and encapsulation efficiency (EE), as well as fresh emulsion droplet size and biopolymer surface amount. Their values, generated from the thirteen experimental runs, are provided in Table 1. As statistical analysis revealed there was no evidence of “lackof-fit” since p values of all parameters were higher than 0.05 (Table 2). Moreover, the coefficient of determination was found to be within 0.8272 and 0.9995 (mean w 0.9517) confirming the desirability of the model to elucidate the relationships between the variables thus allowing an acceptable fitness of response surface models to the experimental data. Moreover, a high value for R2 (adj) demonstrates that non-significant terms have not been included in the model (data not shown). The above results clearly show that the chosen model can satisfactory explain the effect of the two factors, i.e. proteinepolysaccharide mass ratio (A) and b-pinene mass (B) on response parameters, i.e. EY, EL, EE, Gs, D[3,2].
The regression coefficients and their p values determined by the analysis of the data pertaining to the independent and response variables are shown in Table 2. 3.3. Encapsulation yield, loading and efficiency The values of encapsulation yield, determined from the thirteen experimental runs generated by the central composite design, ranged from 14.50 to 47.81 and 1.02 to 42.06% for CNeCMC and WPIeCMC, respectively (Table 1). ANOVA demonstrated that the model was highly significant with R2 of 0.9709 and 0.9842 and fitted well the experimental data with insignificant “lack of fit” (p ¼ 0.091 for CNeCMC and 0.090 for WPIeCMC system) (Table 2). It is apparent that the encapsulation yield was significantly influenced by the positive, in the case of CNeCMC, or negative, in the case of WPIeCMC, linear effect of A (pr/pl ratio) as well as the negative linear effect of B (b-pinene mass) at p < 0.01. There were also significant negative quadratic effects of A and positive quadratic effects of B, while no significant interaction effect was noted for A and B for both systems. In order the correlation between the response of yield (EY%) and the experimental levels of each factor (A, B) to be better understood, response surface plots were created from the model. The 3D graphs that are illustrated in Fig. 2a and Fig. 3a are plotted as a function of the two factors A and B. Fig. 2a depicts that the yield was optimum for small amounts of bpinene and high CNeCMC ratios. On the other hand, in the case of WPIeCMC combination the yield was found to increase, reaching a maximum when A was in the range of 3.5e4 and B was at its lowest level, and decrease with further increase of A to 6.99 (Fig. 3a). The yield of the encapsulation process (%), calculated from the mass ratio of the final product to the initial one (wall and core material) used, actually represents the quantity of biopolymers (wall material) that have interacted and are separated from the serum of the initial emulsion, entrapping at the same time the volatile compound (core material). The amount of non-adsorbed biopolymers and free b-pinene remained “dispersed” in the rest of the emulsion continuous phase. In the case of CNeCMC mixture, the proteinepolysaccharide ratio increase induced a w20% yield increase something that was mostly evident when a small quantity of b-pinene was used (Fig. 2a). In line with this observation, a high amount of biopolymers was adsorbed on the oil droplet surface as
Table 2 Regression coefficients and their p values from analysis of variance for the response variables using coded units.
CNeCMC
WPIeCMC
Gs(Pr)
Gs(CMC)
Responsea
EY
Term
Coef.
p
Coef.
p
Coef.
p
Coef.
p
Const. Ab B A2 B2 A*B Lack of fit R2 Const. A B A2 B2 A*B Lack of fit R2
26.126 2.932 8.596 2.552 4.836 0.853
0.000** 0.004** 0.000** 0.011* 0.000** 0.413 0.091
20.052 2.897 1.666 1.957 3.312 2.223
0.000** 0.004** 0.046* 0.033* 0.003** 0.413 0.240
11.380 4.236 6.502 3.063 0.053 0.115
0.000** 0.000** 0.000** 0.002** 0.937 0.896 0.422
10.308 1.275 17.188 0.647 12.855 0.655
0.000** 0.000** 0.000** 0.022* 0.000** 0.058 0.115
0.9709 21.890 2.761 7.695 9.526 4.279 1.223 0.9842
EE
0.000** 0.003** 0.000** 0.011* 0.000** 0.213 0.090
0.8834 26.376 0.048 5.477 2.780 2.315 2.610 0.8272
EL
0.000** 0.969 0.002** 0.064 0.109 0.161 0.114
0.9649 15.726 4.233 7.148 2.424 1.886 2.260 0.9087
0.000** 0.006** 0.000** 0.074 0.147 0.182 0.073
0.9993 10.366 1.406 20.147 1.939 14.466 3.430 0.9947
0.000** 0.077 0.000** 0.033 0.000** 0.009 0.112
Coef. 5.178 0.000 9.208 0.176 7.639 0.133 0.9993 5.366 3.888 16.624 1.560 10.723 5.310 0.9995
D[3,2] p
Coef.
p
0.000** 0.999 0.000** 0.197 0.000** 0.442 0.338
0.263 0.034 0.049 0.019 0.024 0.033
0.000** 0.004** 0.000** 0.059 0.025* 0.023* 0.069
0.000** 0.000** 0.000** 0.000** 0.000** 0.000** 0.055
0.9156 0.287 0.102 0.012 0.041 0.012 0.082
0.000** 0.000** 0.181 0.002** 0.238 0.000** 0.249
0.9731
* For p < 0.050. ** For p < 0.010. a Responses: EY, encapsulation yield; EE, encapsulation efficiency; EL, encapsulation loading; Gs, adsorbed amount of biopolymers per surface unit; D[3,2], emulsion droplet diameter. b Keys: A: protein/polysaccharide ratio; B: b-pinene mass.
T. Koupantsis et al. / Food Hydrocolloids 37 (2014) 134e142
Fig. 2. Response surface 3D plots for CNeCMC: Effect of protein/polysaccharide mass ratio and b-pinene mass on encapsulation yield (EY %) (A), encapsulation loading (EL %) (B), and encapsulation efficiency (EE %) (C).
was indicated by the high Gs calculated for the respective addition level of the volatile compound (Table 1). This was also supported by the results of statistical analysis where the positive and negative linear effect of A and B on Gs(CN), respectively, is shown (Table 2). The increase of biopolymer ratio (i.e. protein concentration) and core material mass induced the development of either proteine polysaccharide or proteineprotein interactions resulting in limited droplet coverage and enhanced yield values. Furthermore, regarding the WPIeCMC systems, the encapsulation yield was significantly affected by the core material mass, while it was obviously diminished in the case of extreme (very low and very high) mass ratios allowing thus to presume that the interactions between them did not lead to complex formation and precipitation (Fig. 3a). This hypothesis was further supported by the Gs(WPI) decrease upon b-pinene mass increase (p < 0.01). Besides, the encapsulation yield of b-pinene was influenced by the type of wall
139
Fig. 3. Response surface 3D plots for WPIeCMC: Effect of protein/polysaccharide mass ratio and b-pinene mass on encapsulation yield (EY %) (A), encapsulation loading (EL %) (B), and encapsulation efficiency (EE %) (C).
material used, since coacervates prepared with CNeCMC mixture resulted in higher yield than those prepared with the WPIeCMC one. This behaviour could be attributed to the greater affinity of sodium caseinate in comparison to that of WPI to interact with CMC. Encapsulation loading (%), calculated as the ratio of the mass of b-pinene encapsulated in the microcapsules to the mass of the final product, ranged from 2.15 to 25.09 and 1.07 to 30.10% for CNeCMC and WPIeCMC, respectively (Table 1). According to analysis of variance results (Table 2), the model appeared to be adequate, with no significant “lack of fit” and with highly significant R2 (0.9649 and 0.9087). Additionally, EL was significantly affected by the independent variables A and B, with A exhibiting a negative and B a positive linear effect (at p < 0.01). Figs. 2b and 3b show that the higher the core material concentration and the lower the protein:CMC ratio, the higher the encapsulation process loading.
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Probably, low protein content allows for a more satisfactory association between protein and CMC at the interface resulting in the formation of a more “structured” interfacial film than higher protein content. Both wall material concentration as well as core material mass also affects the process in terms of encapsulation efficiency. As Table 1 revealed, EE ranged from 17.8 to 29.02% and 13.05 to 35.54% in the case of CNeCMC and WPIeCMC, respectively. The statistical analysis suggested that the model was appropriate with satisfactory R2 (0.8834 and 0.8272) and no evidence of “lack of fit” since for CNeCMC and WPIeCMC p values were 0.240 and 0.114 (>0.05), respectively (Table 2). Sodium caseinateeCMC microcapsules formation was found to be considerably affected by the mass of core material as well as the biopolymer content. More specifically, EE was significantly influenced by the negative linear effect of A, the positive linear effect of B and the positive quadratic effects of both factors (Table 2, Fig. 2c). This last effect of A and B can be seen by the characteristic curvature of the response surface, especially at values near the middle level of both factors (Fig. 2c). The microcapsules made using biopolymer mass and core material of 3.60 had the lowest encapsulation efficiency (w20%), indicating that relatively greater amount of b-pinene remained in the supernatant instead of being encapsulated in the microcapsules at this ratio. It could be hypothesised that the increase of biopolymer ratio (i.e. protein concentration) induced the development of either proteinepolysaccharide or proteineprotein interactions reducing thereby the ability of protein molecules to reach and become adsorbed at the oil-water interface. In the case of WPIeCMC microcapsules, encapsulation efficiency was appreciably affected by the b-pinene content (positive linear effect at p < 0.01), while the effect of proteinepolysaccharide ratio was found to be no significant (p ¼ 0.969 >> 0.05), i.e. EE was increased when b-pinene content increased. Furthermore, as results of both Tables 1 and 2 revealed, the amount of protein adsorbed on the oil droplet surface (Gs) was greatly decreased by b-pinene mass increase (at p < 0.01), while it was not affected by the proteinepolysacharide ratio variations (p > 0.05). It seems likely that the droplets present in emulsions prepared with high b-pinene addition were not fully covered by biopolymer molecules than those in emulsions prepared with low b-pinene content. This observation is also connected to droplet size increase (positive linear effect of B on D[3,2]) allowing inferring that the increase in the concentration of core material enhanced interactions between the biopolymers used resulting in increased encapsulation efficiency. In general, our results are lower than those reported in the literature something that partially could be attributed to some extent to the fact that no cross-linking agent was used. For example, the efficiency of the encapsulation of propolis by soy protein and pectin as well as capsaicin encapsulation by gelatine-Tween 60 mixture reached a 70% (Nori et al., 2011; Wang, Chen, & Xu, 2008). In addition, the method applied for capsules drying, i.e. freeze-drying, may induced losses of the volatile constituent possibly through capsule disruption at very low temperatures. The encapsulation efficiency depicts the potency of biopolymers firstly in the emulsification and stabilisation of volatile’s droplets and secondly in the protection of the encapsulated aroma compound against losses or oxidation, something that is very important in the food industry for volatile core materials. As the mean droplet size of emulsions was practically the same (D[3,2] ¼ 0.290 and 0.319 mm for CNeCMC and WPIeCMC systems, respectively), the results obtained could be attributed to the differences between the biopolymer materials used and their physicochemical properties (e.g. surface activity, tendency to bind aroma compounds). WPI in combination with CMC appeared to be rather more effective for the encapsulation of b-pinene than sodium caseinate (Figs. 2c and 3c;
Table 1). Other studies have also shown that whey protein was more effective in binding aroma compounds in comparison to sodium caseinate (Hansen & Booker, 1996; Li, Grün, & Fernando, 2000). A similar trend was also observed by Paraskevopoulou, Tsoukala, and Kiosseoglou (2009) who noted that WPI was more effective than CN and Tween 40 in retaining the mastic gum oil volatiles (mainly terpenes) in hydroalcoholic model emulsion systems. The decrease in volatility of the hydrophobic terpenes was attributed to hydrophobic interaction with the central cavity of the protein and more specifically with b-lactoglobulin (b-lg). According to Guichard and Langourieux (2000), the b-lg molecules interact with several aroma compounds (i.e. carbonyl compounds, ionones, hydrocarbons) through two different binding sites (Narayan & Berliner, 1997). Acidic pHs favour these interactions by enhancing the flexibility of the protein molecule and assisting in molecule unfolding and exposure of hidden residues to the surface (Jouenne & Crouzet, 2000). Additionally, as was stated by Paraskevopoulou et al. (2013), the relative amount of the terpenes in the headspace of CMCeWPI-containing systems was significantly lower than in the water phase at both pH values (6.0 and 3.7) studied probably due to hydrophobic interactions with the system components. Upon gelation at pH 3.7, the retention degree was further increased as a result of the entrapment of the aroma compounds inside the developed gel network. 3.4. Morphology of microcapsules The morphology of the produced microcapsules was studied by SEM. The images obtained revealed that the final freeze-dried products exhibited either a compact or a “spongy”-like structure (Fig. 4), which is common for this type of products as stated in the literature (Quispe-Condori, Saldaña, & Temelli, 2011; Saravanan & Panduranga, 2010). In general, the porosity of these structures appears to be influenced by both the biopolymer and b-pinene concentration. Similar findings have been reported by other researchers by using another observation technique, such as X-ray microscope (Laine et al., 2010). In both proteinepolysaccharide combinations, i.e. CNeCMC and WPIeCMC, the surface of freeze dried products with low protein concentration (Fig. 4a and d) exhibited the lower porosity with the droplets coming closer to each other and leaving less space between them, with the second being the denser and more rigid. By increasing the protein concentration, as Fig. 4b for CN and 4e for WPI show, a network was formed in which b-pinene droplets were immobilized. The network of sodium caseinate was formed by chains with rounded edges while that of WPI was flatter with sharper edges. The formation of this network was the result of electrostatic interactions between non-adsorbed proteins and polysaccharides molecules. Furthermore, z-potential values (Fig. 1a and c) corresponding to pH around 2.8 favour electrostatic interactions and bridging flocculation (Dickinson, 2003). In the case of systems that contained the lowest amount of b-pinene (Fig. 4c and f), the same network consisted of elongated chains with branches seemed to be formed. By comparing percentages of encapsulation efficiency (Figs. 2c and 3c) and loading (Figs. 2b and 3b) with the morphology characteristics of the final freeze-dried products it was concluded that both efficiency and loading seemed to be affected by morphology. More specifically, the “spongy”-like structures exhibited lower percentages of encapsulation loading and efficiency than the compact structures. The empty space of “spongy”-like structures resulted in droplets of a larger free surface, thus increasing losses of the volatile constituent. In all cases, the surfaces of the freeze-dried products were smooth and without dents or shrinkages. In general, the microstructure of the final product plays an important role and the presence of cracks and dents on the surface of the dried
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Fig. 4. SEM images of powders produced from different CNeCMC ratios (aec) and b-pinene mass (def): (a) CN/pl ratio 0.2 & b-pinene mass 6.9 g (1000), (b) CN/pl ratio 6.9 & bpinene mass 6.9 (1500), (c) CN/pl ratio 6.9 & b-pinene mass 0.2 (1000), (d) WPI/pl ratio 0.2 & b-pinene mass 6.9 g (1000), (e) WPI/pl ratio 6.9 & b-pinene mass 6.9 (1000) and (f) WPI/pl ratio 6.9 & b-pinene mass 0.2 (1000).
particles affects the retention or losses of aroma compounds (Druaux & Voilley, 1997).
4. Conclusions The lowering of pH to a value near 2.8 of aqueous CNeCMC and WPIeCMC mixtures resulted in the development of coacervatetype complexes that can act as encapsulating agents. As results revealed, most of the produced coacervates could be effective encapsulation systems as well as possible delivery systems in foods. This was more evident in the case of WPIeCMC mixture, possibly due to the enhancement of the protein flexibility and unfolding at acidic pH. In general, protein/polysaccharide ratio increase caused changes in microcapsule morphology, producing a network in which b-pinene droplets were immobilized. The formation of this network was the result of electrostatic interactions between nonadsorbed proteins and polysaccharides molecules. The use of crosslinking agents may improve encapsulation efficiency of milk proteinseCMC coacervates something that is under investigation by our team.
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Flavour encapsulation in milk proteins e CMC coacervate-type complexes T. Koupantsis a, E. Pavlidou b, A. Paraskevopoulou a, * a b
Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece Solid State Physics Section, Physics Department, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2013 Accepted 31 October 2013
Beta-pinene containing microcapsules were prepared by complex coacervation of milk proteins, i.e sodium caseinate (CN) and whey protein isolate (WPI), with carboxymethylcellulose (CMC). Milk proteins e CMC interactions were followed by z-potential measurements, while the initial emulsions were characterised for droplet size and biopolymer amount present at the oil/water interface. Response surface methodology was applied to investigate the effects of encapsulation processing variables, including protein/polysaccharide (pr/pl) ratio and volatile compound’s mass, on encapsulation yield, loading and efficiency as well as the morphological characteristics of the produced microcapsules. The obtained results revealed that it was possible to encapsulate b-pinene with milk proteins and CMC by complex coacervation, while most of the characteristics evaluated were affected by the process variables. Coacervates prepared at the highest pr/pl ratio of 6.99 and b-pinene mass (6.99 g) were the most effective in encapsulating the flavour compound, something that was more evident in the case of WPIeCMC mixture. Additionally, microcapsule structure, evaluated by Scanning Electron Microscopy analysis, was noticeably affected by the protein/polysaccharide ratio being compact when pr/pl was low and “spongy”like when it was high. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Flavour encapsulation Complex coacervation Sodium caseinate Whey protein Carboxymethylcellulose Response surface methodology
1. Introduction Proteinepolysaccharide interactions have found diverse applications in food sector, such as in food colloidal systems formation, fat substitution, protein recovery from fluid by-products and encapsulation (Damianou & Kiosseoglou, 2006; Dickinson, 2003; Kika, Korlos, & Kiosseoglou, 2007; Paraskevopoulou & Kiosseoglou, 2013). Among them, biopolymer-based encapsulation of various ingredients (flavours, vitamins, antioxidants, etc.) offers many advantages in view of protection from losses and environmental factors, unpleasant tastes’ masking, liquid conversion to more convenient solid materials and controlled release (Nori et al., 2011; Qv, Zeng, & Jiang, 2011; Zhang, Pan, & Chung, 2011). In the case of flavour compounds, encapsulation provides an effective way for their stability reinforcement through elimination of problems associated with their exposure to air, light, heat and moisture. It is based on the formation of a coating layer (wall material) to encapsulate flavour droplets or particles (core material). Proteinepolysaccharide complex coacervation is the process
* Corresponding author. Tel.: þ30 2 31 0 997832; fax: þ30 231 0 997847, 997779. E-mail address: [email protected] (A. Paraskevopoulou). 0268-005X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2013.10.031
most commonly used for this purpose. It is, almost entirely, accomplished when two oppositely charged biopolymer molecules are mixed at a pH below the protein isoelectric point (pI) leading to the formation of a complex, i.e. coacervate, which precipitates retaining at the same time the aroma material at the coacervate phase. The success of the process is mainly affected by pH manipulation. More specifically, a soluble proteinepolysaccharide complex is firstly formed followed by complex coacervation and/or precipitation upon further lowering of pH, attributed to the modulation of initial repulsive proteinepolysaccharide interactions into net attractive ones (Paraskevopoulou & Kiosseoglou, 2013). Flavour encapsulation by complex coacervation of proteins with a number of charge-carrying polysaccharides has been the subject of some recently published research papers (Jun-xia, Hai-yan, & Jian, 2011; Leclercq, Harlander, & Reineccius, 2009; Prata, Zanin, Ré, & Grosso, 2008; Yeo, Bellas, Firestone, Langer, & Kohane, 2005). In these studies, various combinations including gelatine or soybean protein isolate with gum arabic, xanthan or pectin have been used for the encapsulation of essential oils or individual flavour compounds. In the present study milk proteins, i.e. sodium caseinate and whey protein isolate, were employed in admixture with carboxymethylcellulose. Milk proteins are commonly used in the food industry for their excellent functional (emulsion preparation and
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stabilisation, water and fat binding, thickening, gelation) and nutritional properties. CN is composed of a mixture of four phosphoproteins, namely as1-, as2-, b- and k-casein. Their highly disordered and hydrophobic character is considered to be responsible for sodium caseinate’s rapid absorption to the oilewater interface during emulsification as well as stabilisation of oil droplets against flocculation and coalescence (Dickinson, 1997). The major component of WPI is b-lactoglobulin, a globular protein known to interact with many aroma compounds due to the existence on the molecule of two independent binding sites (Narayan & Berliner, 1997). Carboxymethylcellulose was selected because it is an anionic polysaccharide that is already widely used as a functional ingredient by the food industry. Its manufacture involves treating cellulose with aqueous sodium hydroxide followed by reaction with monochloroacetic acid. It is water soluble and its solutions exhibit non-Newtonian, pseudoplastic solutions (Zecher & Gerrish, 1999). Milk proteins have been successfully used in combination with polysaccharides such as gum arabic, xanthan, mesquite gum as well as CMC in the formation and stabilisation of food emulsion systems, while, depending on pH, both of them interact with polysaccharides to form soluble or insoluble complex coacervates, nanoparticles or precipitates (Bedie, Turgeon, & Makhlouf, 2008; Benichou, Aserin, Lutz, & Garti, 2007; Ercelebi & Ibanoglu, 2007; Koupantsis & Kiosseoglou, 2009; Liu et al., 2012; Weinbreck, Minor, & de Kruif, 2004; Ye, 2008). More specifically, it was observed that when the pH of an aqueous WPIeCMC solution was steadily reduced to values between 3.5 and 4, a gel-like system was generated upon ageing (Koupantsis & Kiosseoglou, 2009; Paraskevopoulou, Tsioga, Biliaderis, & Kiosseoglou, 2013). The development of the gel structure was primarily attributed to electrostatic interactions between the two biopolymers, while hydrophobic interactions between protein molecules appeared to play a minor role. Additionally, the gel structure formation at low pH enhanced the retention of orange oil aroma compounds when compared with single biopolymers or their mixtures at neutral pH (Paraskevopoulou et al., 2013). In view of the above, this work was conducted to investigate whether complex coacervation of milk proteins with CMC can be used for the encapsulation of flavour compounds, i.e. b-pinene. The initial emulsions were characterised for droplet size and biopolymer adsorption to the oil/water interface, while the microcapsules were characterised for encapsulation efficiency, loading, yield and morphology. Optimisation of the encapsulation process conditions was attempted using response surface methodology where the simultaneous effect of b-pinene amount and protein/polysaccharide mass ratio was investigated. 2. Materials and methods 2.1. Materials Casein sodium salt (from bovine milk) was purchased from Sigma Chemical Co. (Germany). Commercial whey protein isolate (natural, unflavoured, lecithin content <3%) from bovine milk was supplied by Nestlé (Germany). This product contained approximately 98% dry matter, of which 91% was protein. Carboxymethylcellulose sodium salt (with low viscosity of 90e200 mPa at 25 C) and b-pinene (>95%, CAS No.127-91-3) were purchased from Fluka (Switzerland). Analytical grade n-hexane was obtained from Merck (Germany). Sodium azide (Aldrich, Germany) was added to the protein and polysaccharide solutions to prevent microbial growth (final concentration of 0.01% w/v). Hydrochloric acid and sodium hydroxide solutions used for pH adjustment were purchased from Aldrich (Germany). Deionised water was used for the preparation of all solutions and emulsions.
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2.2. Microcapsule preparation 2.2.1. Preparation of biopolymer matrices Individual protein (3.5% w/v) and polysaccharide (0.5% w/v) matrices were initially prepared by dispersing sodium caseinate, whey protein isolate and carboxymethylcellulose in deionised water under mechanical stirring at room temperature for more than 5 h for a full dispersion of the macromolecules. The three solutions were then used to prepare the emulsions. Blends containing 0.25% (w/v) CMC and different amounts of CN or WPI were obtained by mechanical mixing at 300 rpm (IKA, Malaysia) for at least 30 min to assure the complete formation of biopolymer mixtures (Table 1). 2.2.2. Formation of o/w emulsions Oil-in-water emulsions were prepared at room temperature by mixing appropriate amount of b-pinene with 200 mL protein/polysaccharide solution with the aid of a mechanical stirrer at 600 rpm (IKA, Malaysia), followed by homogenisation at 30 106 Pa (4 passes) using an APV-2000 pressure homogenizer (APV Systems, Denmark). The produced emulsions showed a final composition, which is shown in Table 1. Sodium azide (0.01% w/v) was added to all emulsions in order to prevent microbial growth. The losses during emulsion preparation were found to be very limited (<2%) and were mainly due to emulsion quantity remaining in the glassware and apparatus used for its preparation as well as volatilisation in the air. 2.2.3. Preparation of microcapsules Four hundred millilitres of deionised water was slowly poured into a beaker containing the final emulsion (w200 mL) and the pH was adjusted to 2.8 with hydrochloric acid solution (1 mol/L) under continuous agitation with the aid of a magnetic stirrer (300 rpm). During the coacervation process the temperature was kept at around 23 C and the suspension was kept at w4 C overnight to allow the coacervation process to be completed (w20 h) (Lamprecht, Schafer, & Lehr, 2001; Leclercq et al., 2009; Zhang et al., 2011). The precipitates were first washed by decanting with deionised water and then isolated by centrifugation (4000 rpm, 10 min), frozen and lyophilised for 5e6 h by using a laboratoryscale freeze dryer (Alpha 1-2, Christ, Germany). The resulting microcapsules were stored at 4 C in a desiccator for further analyses. 2.3. Emulsion droplet size measurement Droplet size distributions and average droplet diameter D[3,2] of the freshly prepared emulsions (before pH adjustment) was determined by the laser diffraction technique and the aid of a Malvern Mastersizer 2000 (Malvern Instruments, Worcestershire, UK) (b-pinene and water refractive index: 1.4650 and 1.33, respectively; Absorption: 0.002) (Reich & Sanhueza, 1993). Each sample was analysed in triplicate and each replicate was measured twice to have the average particle size. 2.4. z-Potential measurements of protein and polysaccharide solutions The electrical charge of CN, WPI and CMC solutions and their mixtures was measured using a Zetasizer ZEN2600 (Malvern Instruments, Worcestershire, UK). For this reason, stock solutions of each biopolymer were first prepared (0.05% w/v). Mixed solutions containing CMC and CN or WPI at various ratios (Table 1) were obtained by thorough mixing appropriate amounts of the initial stock solutions. The individual as well as the mixed solutions were then diluted to a concentration of approximately 0.02% (w/v) with deionised water and, after adjustment to the suitable pH, were
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Table 1 Experimental design (conditions and responses) for encapsulation yield (EY%), encapsulation efficiency (EE%), encapsulation loading (EL%), adsorbed amount of biopolymers per surface unit (Gs) and emulsion droplet diameter (D[3,2]). xPr:yPla
Run
Factor A Pr/Pl ratio
Factor B
b-pinene (g)
CNeCMC EY (%)
WPIeCMC EE (%)
EL (%)
Gs (CN)
Gs (CMC)
EY (%)
(mg/m2)
D[3,2] (mm)
10.05 38.50 39.73 61.10 10.23 10.54 7.55 9.87 10.85 10.97 7.35 11.23 3.70
5.28 21.95 21.67 33.59 5.41 5.13 3.87 4.69 5.38 4.70 4.68 7.05 3.62
0.256 0.332 0.213 0.221 0.245 0.273 0.341 0.265 0.278 0.247 0.362 0.408 0.330
20.80 29.17 18.41 42.06 22.29 23.56 6.34 21.90 20.90 1.02 4.88 19.06 12.21
(mg/m2) 1 2 3 4 5 6 7 8 9 10 11 12 13 a
52:100 17:100 86:100 52:100 52:100 52:100 86:100 52:100 52:100 100:100 3:100 52:100 17:100
3.60 1.20 6.00 3.60 3.60 3.60 6.00 3.60 3.60 6.99 0.21 3.60 1.20
3.60 1.20 1.20 0.21 3.60 3.60 6.00 3.60 3.60 3.60 3.60 6.99 6.00
25.20 34.96 39.32 47.81 26.20 25.44 20.37 25.54 28.25 27.33 14.50 23.57 19.42
17.80 26.80 17.83 26.19 19.01 21.31 28.17 21.55 20.59 19.03 29.02 27.28 28.25
10.86 11.09 4.55 2.15 8.88 12.56 17.79 11.60 13.00 10.05 25.09 20.53 23.87
EE (%)
EL (%)
Gs (WPI)
Gs (CMC)
(mg/m2)
D[3,2] (mm)
10.63 47.15 36.05 69.80 9.31 11.83 5.37 8.79 11.27 5.57 7.53 8.92 2.75
5.40 43.56 24.65 50.61 5.69 5.54 2.60 5.15 5.05 3.23 13.50 2.77 0.27
0.260 0.560 0.165 0.310 0.288 0.300 0.291 0.307 0.281 0.240 0.490 0.299 0.357
(mg/m2) 24.95 22.00 22.07 13.05 26.11 23.57 36.90 28.67 28.58 28.33 35.54 30.44 26.39
13.39 13.67 5.64 1.07 15.93 14.38 23.31 17.49 17.44 11.12 30.10 22.91 22.30
“x” and “y”: volumes of initial protein (3.5% w/v) and polysaccharide (0.5% w/v) solutions, respectively.
placed into the measurement cell of the instrument. The measurements were converted to z-potential values by applying the Smoluchowski mathematical model and are reported as average of three replicates, each of which was measured three times. The temperature was kept constant at 22 C.
after it was taken out of the freeze, it was centrifuged at 4000 rpm for 40 min, placed in a water bath of 50 C for 40 min and recentrifuged at 4000 rpm for 10 min until complete phase separation. The organic phase was collected and was subsequently stored in screw-capped glass vials at 18 C until further analysis. Each powdered sample was extracted in triplicate.
2.5. Protein and polysaccharide surface amount determination The amount of protein and polysaccharide adsorbed at the surface of the emulsion droplets was determined from the difference between the initial amount of biopolymers used and that remained in the serum after the coacervation was completed. Total protein and polysaccharide content was determined by applying the FolineCiocalteu (Lowry, Rosebrough, Farr, & Randall, 1951) and phenolesulphuric method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), respectively. More specifically, for the determination of the surface protein (or polysaccharide) concentration Gs (mg/m2), the following equations were used (Walstra, 1983):
SSA ¼
6 D½3;2
ST ¼ SSA$V
GS ¼
GT
2.7. GC-FID analyses The analyses were accomplished with an Agilent 6890A gas chromatograph equipped with a split-splitless injector and a flame ionization detector (FID). The samples were analysed on a HP-FFAP column (25 m 0.20 mm i.d., film thickness 0.30 mm; Agilent Technologies). The carrier gas was helium at a constant flow rate of 1 mL/min. Samples (2 mL) were injected manually into the GC in split mode with 1:100 ratio. Injector and detector were both kept at 230 C. The temperature program was 40 C for 2 min, raised to 100 C at 10 C/min, then raised to 230 C at 30 C/min and held for 1 min. The obtained peak areas were converted to concentrations using a calibration curve (y ¼ 0.3732x þ 44.656). For its construction, b-pinene solutions were prepared in hexane at ten different concentration levels (0.10e6.24 g/L) and analysed five times applying the same conditions as described previously for the samples. Linear correlation coefficient was found equal to 0.9985. All analyses were performed at least three times (CV < 3%).
ST
where, SSA is the specific surface area (m2/mL), D[3,2] is the volumeesurface mean droplet diameter (mm), ST is the total surface of oil droplets (m2), V is the volume of b-pinene (mL) and GT is the total biopolymer at the surface (mg) calculated by the difference between the biopolymer amount used initially and that found in the serum. 2.6. Extraction of encapsulated b-pinene Beta-pinene was extracted as described by other researchers with slight modifications (Soottitantawat et al., 2004, 2005; Zhang et al., 2011). Five mL of deionised water was added to 0.1 g of the freeze-dried powder in a screw-capped glass tube, followed by 2 mL hexane (pH was adjusted to 7.0 by using a 0.1 mol/L sodium hydroxide solution). The resulting solution was vortexed for 1 min at room temperature and stored at 18 C overnight. Immediately
2.8. Microcapsules characterisation The b-pinene containing particles were characterised with respect to yield, encapsulation efficiency, encapsulation loading and morphology. The three encapsulation monitoring parameters were calculated as follows:
Encapsulation Yield ð%Þ ¼
Mmicrocapsules $100% Mtotal
Encapsulation Loading ð%Þ ¼
Encapsulation Efficiency ð%Þ ¼
Mbpinene encapsulated $100% Mmicrocapsules Mbpinene Mbpinene to
encapsulated be encapsulated
$100%
T. Koupantsis et al. / Food Hydrocolloids 37 (2014) 134e142
where Mmicrocapsules is the mass of the final product, Mtotal is the mass of initial material used (wall and core), Mb-pinene encapsulated is the actual amount of b-pinene encapsulated and Mb-pinene to be encapsulated the theoretical mass of b-pinene to be encapsulated. The morphology of the microcapsules was performed by Scanning Electron Microscopy (SEM), using a JEOL 840A SEM operated at 20 kV equipped with an INCA 300 EDS analyzer. The dried samples under investigation were placed on sample holders and coated by carbon, using a JEE-4X vacuum evaporator, in order to achieve electrical conductivity.
137
A
2.9. Experimental design A rotatable central composite design was used to perform the tests for the microencapsulation of b-pinene, considering two factors (independent variables): mass of b-pinene (0.21e6.99 g) and the mass ratio of protein (CN or WPI) and polysaccharide (CMC) (0.21e6.99 g). The experimental plan consisted of 13 trials (5 for the central point replicated for the estimation of error) and the independent variables were studied at five different levels (Table 1). Central point conditions were 3.6 g b-pinene and 3.6 protein/ polysaccharide mass ratio. The following polynomial equation was fitted to data:
B
y ¼ b0 þ b1 A þ b2 B þ b11 A2 þ b22 B2 þ b12 AB where, bn are constant regression coefficients, y is the response variable (encapsulation efficiency, encapsulation loading or encapsulation yield) and A and B are the coded independent variables corresponding to protein/polysaccharide mass ratio and bpinene mass, respectively. The statistical analysis of the data was performed by using MINITABÔ Statistical Software and the level of significance was 95%. The quality of fit of the model was evaluated on the basis of coefficients of determination (R2), the significance of each parameter (through calculated p values) and the “lack of fit”.
C
3. Results and discussion 3.1. pH selection In this study, because of the use of the anionic polysaccharide carboxymethylcellulose along with sodium caseinate or whey protein isolate, complex coacervation was found to be favoured at a pH around 2.8, significantly below the protein isoelectric point (w4.6 for CN and 4.1e5.2 for WPI) (Koupantsis & Kiosseoglou, 2009; Liu et al., 2012). For the selection of this pH, z-potential values (Fig. 1a) as well the mass of the precipitated material and the turbidity of the supernatant (data not shown) at various pH levels (range 2.5e7.3) were taken into account (Nori et al., 2011). More specifically, as Fig. 1a reveals, at pH 2.8 there is enough charge in order interactions between protein and polysaccharide to take place. In addition, at this pH value the supernatant was almost a clear solution while the larger quantity of precipitate was observed. Generally, in the case of anionic polysaccharides the optimum pH for coacervation is below the protein isoelectric point (pI) which is explained by the development of intense attractive electrostatic forces between the biopolymers (Koupantsis & Kiosseoglou, 2009; Paraskevopoulou et al., 2013; Paraskevopoulou & Kiosseoglou, 2013). Generally, at a pH close to neutrality (w6.8) interactions between protein and polysaccharide molecules were not favoured as both of them carry a net negative charge (Fig. 1b). However, the existence of weak interactions has been reported by several researchers for various proteinepolysaccharide pairs, such as BSAeicarrageenan, WPIexanthan, WPC or WPIeCMC (Benichou et al.,
Fig. 1. z-potential of biopolymer solutions as affected by the pH (A) and the protein/ polysaccharide ratio at pH 6.8 (B) and at pH 2.8 (C). Keys: CN; WPI; CMC; CNe CMC mixture; WPIeCMC mixture.
2007; Koupantsis & Kiosseoglou, 2009; Paraskevopoulou et al., 2013). The emulsions prepared with the use of both studied combinations (CNeCMC and WPIeCMC) exhibited monomodal distribution and similar droplet mean diameter characteristics. As pH was decreased to a value of 2.8, the protein molecules became positively charged while CMC molecules less negatively charged (Fig. 1a). This suggests that, upon lowering of pH to a value below enough the isoelectric point of the proteins, electrostatic interactions between positively and negatively charges located on the protein and CMC chains, respectively, took place which led to the formation of insoluble complexes (Paraskevopoulou et al., 2013; Schmitt & Turgeon, 2011; Ye, 2008). As Fig. 1c shows, at this pH the biopolymers possessed sufficient electric charge that allowed the molecules to interact with each other. Moreover, the biopolymer mixing ratio is considered to be crucial in controlling system’s charge and thus the extent of interactions and aggregation
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T. Koupantsis et al. / Food Hydrocolloids 37 (2014) 134e142
phenomena between protein and polysaccharide (Ye, 2008). Actually, depending on the protein/CMC ratio, these dispersed proteine polysaccharide coacervates aggregated and formed clusters that grew in size and, in most of the cases, precipitated (visually observed). This is in agreement with the reports of many researchers for various proteinepolysaccharide systems, including soybean protein isolateepectin and gelatineegum arabic, who indicated that at a pH between the protein pI and polysaccharide pK, this interaction, which is mainly electrostatic in nature, usually results in biopolymer coacervation and, upon further lowering of pH, in insoluble complex formation and precipitation (Nori et al., 2011; Leclercq et al., 2009; Zhang et al., 2011). In addition to electrostatic interactions, physical interactions, such as hydrogen and/ or hydrophobic ones, may play a role in proteinepolysaccharide coacervation and complex formation (Tolstoguzov, 1986). 3.2. Response surface methodology The process of b-pinene-containing microcapsules production was standardised for the maximum encapsulation by using response surface methodology. As has already been mentioned, two different biopolymer combinations were tested, i.e. CMC was combined either with CN or WPI by using complex coacervation. Three parameters were applied for the characterisation of dried microcapsules, namely yield of the encapsulation procedure (EY), encapsulation loading of b-pinene (EL) in the final product and encapsulation efficiency (EE), as well as fresh emulsion droplet size and biopolymer surface amount. Their values, generated from the thirteen experimental runs, are provided in Table 1. As statistical analysis revealed there was no evidence of “lackof-fit” since p values of all parameters were higher than 0.05 (Table 2). Moreover, the coefficient of determination was found to be within 0.8272 and 0.9995 (mean w 0.9517) confirming the desirability of the model to elucidate the relationships between the variables thus allowing an acceptable fitness of response surface models to the experimental data. Moreover, a high value for R2 (adj) demonstrates that non-significant terms have not been included in the model (data not shown). The above results clearly show that the chosen model can satisfactory explain the effect of the two factors, i.e. proteinepolysaccharide mass ratio (A) and b-pinene mass (B) on response parameters, i.e. EY, EL, EE, Gs, D[3,2].
The regression coefficients and their p values determined by the analysis of the data pertaining to the independent and response variables are shown in Table 2. 3.3. Encapsulation yield, loading and efficiency The values of encapsulation yield, determined from the thirteen experimental runs generated by the central composite design, ranged from 14.50 to 47.81 and 1.02 to 42.06% for CNeCMC and WPIeCMC, respectively (Table 1). ANOVA demonstrated that the model was highly significant with R2 of 0.9709 and 0.9842 and fitted well the experimental data with insignificant “lack of fit” (p ¼ 0.091 for CNeCMC and 0.090 for WPIeCMC system) (Table 2). It is apparent that the encapsulation yield was significantly influenced by the positive, in the case of CNeCMC, or negative, in the case of WPIeCMC, linear effect of A (pr/pl ratio) as well as the negative linear effect of B (b-pinene mass) at p < 0.01. There were also significant negative quadratic effects of A and positive quadratic effects of B, while no significant interaction effect was noted for A and B for both systems. In order the correlation between the response of yield (EY%) and the experimental levels of each factor (A, B) to be better understood, response surface plots were created from the model. The 3D graphs that are illustrated in Fig. 2a and Fig. 3a are plotted as a function of the two factors A and B. Fig. 2a depicts that the yield was optimum for small amounts of bpinene and high CNeCMC ratios. On the other hand, in the case of WPIeCMC combination the yield was found to increase, reaching a maximum when A was in the range of 3.5e4 and B was at its lowest level, and decrease with further increase of A to 6.99 (Fig. 3a). The yield of the encapsulation process (%), calculated from the mass ratio of the final product to the initial one (wall and core material) used, actually represents the quantity of biopolymers (wall material) that have interacted and are separated from the serum of the initial emulsion, entrapping at the same time the volatile compound (core material). The amount of non-adsorbed biopolymers and free b-pinene remained “dispersed” in the rest of the emulsion continuous phase. In the case of CNeCMC mixture, the proteinepolysaccharide ratio increase induced a w20% yield increase something that was mostly evident when a small quantity of b-pinene was used (Fig. 2a). In line with this observation, a high amount of biopolymers was adsorbed on the oil droplet surface as
Table 2 Regression coefficients and their p values from analysis of variance for the response variables using coded units.
CNeCMC
WPIeCMC
Gs(Pr)
Gs(CMC)
Responsea
EY
Term
Coef.
p
Coef.
p
Coef.
p
Coef.
p
Const. Ab B A2 B2 A*B Lack of fit R2 Const. A B A2 B2 A*B Lack of fit R2
26.126 2.932 8.596 2.552 4.836 0.853
0.000** 0.004** 0.000** 0.011* 0.000** 0.413 0.091
20.052 2.897 1.666 1.957 3.312 2.223
0.000** 0.004** 0.046* 0.033* 0.003** 0.413 0.240
11.380 4.236 6.502 3.063 0.053 0.115
0.000** 0.000** 0.000** 0.002** 0.937 0.896 0.422
10.308 1.275 17.188 0.647 12.855 0.655
0.000** 0.000** 0.000** 0.022* 0.000** 0.058 0.115
0.9709 21.890 2.761 7.695 9.526 4.279 1.223 0.9842
EE
0.000** 0.003** 0.000** 0.011* 0.000** 0.213 0.090
0.8834 26.376 0.048 5.477 2.780 2.315 2.610 0.8272
EL
0.000** 0.969 0.002** 0.064 0.109 0.161 0.114
0.9649 15.726 4.233 7.148 2.424 1.886 2.260 0.9087
0.000** 0.006** 0.000** 0.074 0.147 0.182 0.073
0.9993 10.366 1.406 20.147 1.939 14.466 3.430 0.9947
0.000** 0.077 0.000** 0.033 0.000** 0.009 0.112
Coef. 5.178 0.000 9.208 0.176 7.639 0.133 0.9993 5.366 3.888 16.624 1.560 10.723 5.310 0.9995
D[3,2] p
Coef.
p
0.000** 0.999 0.000** 0.197 0.000** 0.442 0.338
0.263 0.034 0.049 0.019 0.024 0.033
0.000** 0.004** 0.000** 0.059 0.025* 0.023* 0.069
0.000** 0.000** 0.000** 0.000** 0.000** 0.000** 0.055
0.9156 0.287 0.102 0.012 0.041 0.012 0.082
0.000** 0.000** 0.181 0.002** 0.238 0.000** 0.249
0.9731
* For p < 0.050. ** For p < 0.010. a Responses: EY, encapsulation yield; EE, encapsulation efficiency; EL, encapsulation loading; Gs, adsorbed amount of biopolymers per surface unit; D[3,2], emulsion droplet diameter. b Keys: A: protein/polysaccharide ratio; B: b-pinene mass.
T. Koupantsis et al. / Food Hydrocolloids 37 (2014) 134e142
Fig. 2. Response surface 3D plots for CNeCMC: Effect of protein/polysaccharide mass ratio and b-pinene mass on encapsulation yield (EY %) (A), encapsulation loading (EL %) (B), and encapsulation efficiency (EE %) (C).
was indicated by the high Gs calculated for the respective addition level of the volatile compound (Table 1). This was also supported by the results of statistical analysis where the positive and negative linear effect of A and B on Gs(CN), respectively, is shown (Table 2). The increase of biopolymer ratio (i.e. protein concentration) and core material mass induced the development of either proteine polysaccharide or proteineprotein interactions resulting in limited droplet coverage and enhanced yield values. Furthermore, regarding the WPIeCMC systems, the encapsulation yield was significantly affected by the core material mass, while it was obviously diminished in the case of extreme (very low and very high) mass ratios allowing thus to presume that the interactions between them did not lead to complex formation and precipitation (Fig. 3a). This hypothesis was further supported by the Gs(WPI) decrease upon b-pinene mass increase (p < 0.01). Besides, the encapsulation yield of b-pinene was influenced by the type of wall
139
Fig. 3. Response surface 3D plots for WPIeCMC: Effect of protein/polysaccharide mass ratio and b-pinene mass on encapsulation yield (EY %) (A), encapsulation loading (EL %) (B), and encapsulation efficiency (EE %) (C).
material used, since coacervates prepared with CNeCMC mixture resulted in higher yield than those prepared with the WPIeCMC one. This behaviour could be attributed to the greater affinity of sodium caseinate in comparison to that of WPI to interact with CMC. Encapsulation loading (%), calculated as the ratio of the mass of b-pinene encapsulated in the microcapsules to the mass of the final product, ranged from 2.15 to 25.09 and 1.07 to 30.10% for CNeCMC and WPIeCMC, respectively (Table 1). According to analysis of variance results (Table 2), the model appeared to be adequate, with no significant “lack of fit” and with highly significant R2 (0.9649 and 0.9087). Additionally, EL was significantly affected by the independent variables A and B, with A exhibiting a negative and B a positive linear effect (at p < 0.01). Figs. 2b and 3b show that the higher the core material concentration and the lower the protein:CMC ratio, the higher the encapsulation process loading.
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Probably, low protein content allows for a more satisfactory association between protein and CMC at the interface resulting in the formation of a more “structured” interfacial film than higher protein content. Both wall material concentration as well as core material mass also affects the process in terms of encapsulation efficiency. As Table 1 revealed, EE ranged from 17.8 to 29.02% and 13.05 to 35.54% in the case of CNeCMC and WPIeCMC, respectively. The statistical analysis suggested that the model was appropriate with satisfactory R2 (0.8834 and 0.8272) and no evidence of “lack of fit” since for CNeCMC and WPIeCMC p values were 0.240 and 0.114 (>0.05), respectively (Table 2). Sodium caseinateeCMC microcapsules formation was found to be considerably affected by the mass of core material as well as the biopolymer content. More specifically, EE was significantly influenced by the negative linear effect of A, the positive linear effect of B and the positive quadratic effects of both factors (Table 2, Fig. 2c). This last effect of A and B can be seen by the characteristic curvature of the response surface, especially at values near the middle level of both factors (Fig. 2c). The microcapsules made using biopolymer mass and core material of 3.60 had the lowest encapsulation efficiency (w20%), indicating that relatively greater amount of b-pinene remained in the supernatant instead of being encapsulated in the microcapsules at this ratio. It could be hypothesised that the increase of biopolymer ratio (i.e. protein concentration) induced the development of either proteinepolysaccharide or proteineprotein interactions reducing thereby the ability of protein molecules to reach and become adsorbed at the oil-water interface. In the case of WPIeCMC microcapsules, encapsulation efficiency was appreciably affected by the b-pinene content (positive linear effect at p < 0.01), while the effect of proteinepolysaccharide ratio was found to be no significant (p ¼ 0.969 >> 0.05), i.e. EE was increased when b-pinene content increased. Furthermore, as results of both Tables 1 and 2 revealed, the amount of protein adsorbed on the oil droplet surface (Gs) was greatly decreased by b-pinene mass increase (at p < 0.01), while it was not affected by the proteinepolysacharide ratio variations (p > 0.05). It seems likely that the droplets present in emulsions prepared with high b-pinene addition were not fully covered by biopolymer molecules than those in emulsions prepared with low b-pinene content. This observation is also connected to droplet size increase (positive linear effect of B on D[3,2]) allowing inferring that the increase in the concentration of core material enhanced interactions between the biopolymers used resulting in increased encapsulation efficiency. In general, our results are lower than those reported in the literature something that partially could be attributed to some extent to the fact that no cross-linking agent was used. For example, the efficiency of the encapsulation of propolis by soy protein and pectin as well as capsaicin encapsulation by gelatine-Tween 60 mixture reached a 70% (Nori et al., 2011; Wang, Chen, & Xu, 2008). In addition, the method applied for capsules drying, i.e. freeze-drying, may induced losses of the volatile constituent possibly through capsule disruption at very low temperatures. The encapsulation efficiency depicts the potency of biopolymers firstly in the emulsification and stabilisation of volatile’s droplets and secondly in the protection of the encapsulated aroma compound against losses or oxidation, something that is very important in the food industry for volatile core materials. As the mean droplet size of emulsions was practically the same (D[3,2] ¼ 0.290 and 0.319 mm for CNeCMC and WPIeCMC systems, respectively), the results obtained could be attributed to the differences between the biopolymer materials used and their physicochemical properties (e.g. surface activity, tendency to bind aroma compounds). WPI in combination with CMC appeared to be rather more effective for the encapsulation of b-pinene than sodium caseinate (Figs. 2c and 3c;
Table 1). Other studies have also shown that whey protein was more effective in binding aroma compounds in comparison to sodium caseinate (Hansen & Booker, 1996; Li, Grün, & Fernando, 2000). A similar trend was also observed by Paraskevopoulou, Tsoukala, and Kiosseoglou (2009) who noted that WPI was more effective than CN and Tween 40 in retaining the mastic gum oil volatiles (mainly terpenes) in hydroalcoholic model emulsion systems. The decrease in volatility of the hydrophobic terpenes was attributed to hydrophobic interaction with the central cavity of the protein and more specifically with b-lactoglobulin (b-lg). According to Guichard and Langourieux (2000), the b-lg molecules interact with several aroma compounds (i.e. carbonyl compounds, ionones, hydrocarbons) through two different binding sites (Narayan & Berliner, 1997). Acidic pHs favour these interactions by enhancing the flexibility of the protein molecule and assisting in molecule unfolding and exposure of hidden residues to the surface (Jouenne & Crouzet, 2000). Additionally, as was stated by Paraskevopoulou et al. (2013), the relative amount of the terpenes in the headspace of CMCeWPI-containing systems was significantly lower than in the water phase at both pH values (6.0 and 3.7) studied probably due to hydrophobic interactions with the system components. Upon gelation at pH 3.7, the retention degree was further increased as a result of the entrapment of the aroma compounds inside the developed gel network. 3.4. Morphology of microcapsules The morphology of the produced microcapsules was studied by SEM. The images obtained revealed that the final freeze-dried products exhibited either a compact or a “spongy”-like structure (Fig. 4), which is common for this type of products as stated in the literature (Quispe-Condori, Saldaña, & Temelli, 2011; Saravanan & Panduranga, 2010). In general, the porosity of these structures appears to be influenced by both the biopolymer and b-pinene concentration. Similar findings have been reported by other researchers by using another observation technique, such as X-ray microscope (Laine et al., 2010). In both proteinepolysaccharide combinations, i.e. CNeCMC and WPIeCMC, the surface of freeze dried products with low protein concentration (Fig. 4a and d) exhibited the lower porosity with the droplets coming closer to each other and leaving less space between them, with the second being the denser and more rigid. By increasing the protein concentration, as Fig. 4b for CN and 4e for WPI show, a network was formed in which b-pinene droplets were immobilized. The network of sodium caseinate was formed by chains with rounded edges while that of WPI was flatter with sharper edges. The formation of this network was the result of electrostatic interactions between non-adsorbed proteins and polysaccharides molecules. Furthermore, z-potential values (Fig. 1a and c) corresponding to pH around 2.8 favour electrostatic interactions and bridging flocculation (Dickinson, 2003). In the case of systems that contained the lowest amount of b-pinene (Fig. 4c and f), the same network consisted of elongated chains with branches seemed to be formed. By comparing percentages of encapsulation efficiency (Figs. 2c and 3c) and loading (Figs. 2b and 3b) with the morphology characteristics of the final freeze-dried products it was concluded that both efficiency and loading seemed to be affected by morphology. More specifically, the “spongy”-like structures exhibited lower percentages of encapsulation loading and efficiency than the compact structures. The empty space of “spongy”-like structures resulted in droplets of a larger free surface, thus increasing losses of the volatile constituent. In all cases, the surfaces of the freeze-dried products were smooth and without dents or shrinkages. In general, the microstructure of the final product plays an important role and the presence of cracks and dents on the surface of the dried
T. Koupantsis et al. / Food Hydrocolloids 37 (2014) 134e142
141
Fig. 4. SEM images of powders produced from different CNeCMC ratios (aec) and b-pinene mass (def): (a) CN/pl ratio 0.2 & b-pinene mass 6.9 g (1000), (b) CN/pl ratio 6.9 & bpinene mass 6.9 (1500), (c) CN/pl ratio 6.9 & b-pinene mass 0.2 (1000), (d) WPI/pl ratio 0.2 & b-pinene mass 6.9 g (1000), (e) WPI/pl ratio 6.9 & b-pinene mass 6.9 (1000) and (f) WPI/pl ratio 6.9 & b-pinene mass 0.2 (1000).
particles affects the retention or losses of aroma compounds (Druaux & Voilley, 1997).
4. Conclusions The lowering of pH to a value near 2.8 of aqueous CNeCMC and WPIeCMC mixtures resulted in the development of coacervatetype complexes that can act as encapsulating agents. As results revealed, most of the produced coacervates could be effective encapsulation systems as well as possible delivery systems in foods. This was more evident in the case of WPIeCMC mixture, possibly due to the enhancement of the protein flexibility and unfolding at acidic pH. In general, protein/polysaccharide ratio increase caused changes in microcapsule morphology, producing a network in which b-pinene droplets were immobilized. The formation of this network was the result of electrostatic interactions between nonadsorbed proteins and polysaccharides molecules. The use of crosslinking agents may improve encapsulation efficiency of milk proteinseCMC coacervates something that is under investigation by our team.
Acknowledgements The authors wish to thank Dr. F. Mantzouridou for advice regarding the application of response surface methodology as well as Dr. C. Malhiac and Dr. N. Hucher (University of Le Havre, France) for their help in the determination of z-potential. References Bedie, G. K., Turgeon, S. L., & Makhlouf, J. (2008). Formation of native whey protein isolate-low methoxyl pectin complexes as a matrix for hydro-soluble food ingredient entrapment in acidic foods. Food Hydrocolloids, 22, 836e844.
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